Altered innate immune profile in blood of systemic mastocytosis patients

Abstract Background Mast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM. Methods Overall, we studied 115 SM patients ‐ 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)‐ and 32 healthy donors (HD). Spontaneous and in vitro‐stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated. Results SM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1β, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ‐stimulated blood monocytes to produce IL1β and TGFβ. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non‐classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL+ DC was specifically encountered in BMM cases. Conclusion These results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.

MC are myeloid cells that play key roles in the innate/inflammatory immune response. 11 Once activated, MC interact with other immune cells either by direct cell-to-cell contact or via release of mediators that act on surrounding immune cells, including monocytes, macrophages, dendritic cells (DC), T-cells and endothelial cells. 12 MC-released serine proteases such as α-tryptase, cleave receptors -e.g. PAR2 (protease-activated receptor-2) and EMR2 (epidermal growth factor-like module-containing mucin-like hormone receptor-like 2)-expressed on the surface membrane of human monocytes and macrophages, leading to their downstream activation. 13,14 In parallel, other MC mediators such as tumour necrosis factor-alpha (TNFα) and prostaglandin D2 (PGD2) also activate macrophages and instruct DC to polarize TCD4 + cells toward a Thelper 2 (Th2) response, respectively. 15,16 Those effects of MC mediators on surrounding immune cells are further enhanced by activated MC-derived exosomes which have been shown to induce in vitro phenotypic and functional maturation of DC, 17 and directly promote activation, proliferation and cytokine secretion -e.g., interleukin (IL)-22, interferon (IFN)-γ and TNFα-by TCD4 + cells following HLA class-II and CD40-CD154-mediated signaling. 18 Based on this immunomodulatory role of MC, it might be hypothesized that constitutive activation of KIT in SM MC, may lead to an altered distribution and/or functional behaviour of other immune cells, with further impact on dysregulated immune responses and disease behavior. [19][20][21] In this regard, increased evidences support an altered innate and adaptative immune response in SM. Thus, abnormally decreased TCD8 + and NK-cell counts together with increased levels of circulating type II innate lymphoid cells (ILC2) have been reported in ISM, 22,23 in parallel to an increase in plasma of IL6 and other inflammatory cytokines, which would further contribute to an enhanced activation and accumulation of clonal MC in SM. 24 Despite these findings, the major (cell) source of these cytokines and their pathogenic role in the outcome of SM, remain poorly understood.
Thus, Tobio et al. 25 26,27 Overall, the latter findings suggest that immune cells other than MC, capable of producing IL6 and other inflammatory cytokines, might be key in enhancing immune dysregulation in SM. 27 Among candidate cells, blood monocytes, tissue macrophages and DC are included.
Here we investigated the distribution and functional behavior of different populations of blood monocytes and DC in a large series of 115 SM patients. Our ultimate goal was to gain insight into the potential alterations of innate myeloid cells in blood of patients with distinct diagnostic subtypes of SM.  Table 1 in Supplementary Information and their distribution per each set of assays performed is detailed in Table 2

| Spontaneous and in vitro-stimulated cytokine production by blood monocytes
For ex vivo cytokine production assays, blood was diluted 1:1 (vol:

| Quantification of cytokine levels in plasma
Soluble TNFα, IL1β, IL6, IL8, IL10 and IL12 levels were assessed in plasma using the Cytometric Bead Array (CBA) immunoassay system (BD) and the Human Inflammatory Cytokine Kit (BD), strictly following the recommendations of the manufacturer. Afterward, a minimum of 3000 events per bead population was measured in a FACSCanto II flow cytometer (BD) using FACSDiva™. For data analysis the FCAP Array software (v3.0; BD) was used.

| Flow cytometric identification and enumeration of blood monocyte and dendritic cell populations
The distribution of distinct subsets of monocytes and DC was investigated in K3-EDTA-anticoagulated fresh (<24 h) blood. For this purpose, 10 7 cells/sample were stained with the Euroflow 14-color  Table 3) using the EuroFlow Bulk Lysis standard operating procedure for staining of cell surface membrane markers-only (www.euroflow.org), as previously described. [28][29][30] In a subset of SM patients and HD, expression of EMR2 (CD312) and the IL8-receptor (CD182) on different subsets of stained blood monocytes and DC were also evaluated ( Table 3 in    Armonk, NY) was used. p-values < 0.05 were considered to be associated with statistical significance.

| Spontaneous ex vivo cytokine production by blood monocytes and cytokine plasma levels in SM
Under baseline ex vivo conditions, blood monocytes from SM patients showed a significantly (P < 0.001) increased (vs. HD) spontaneous production of IL1β, IL6, IL8, TNFα, and to a less extent also IL10 (P = 0.036), with higher numbers of cytokine-producing monocytes ( Figure 1A,B). Of note, increased numbers of IL6 + , IL8 + and TNFα + monocytes in blood after short-term in vitro culture To determine whether the increased spontaneous production of inflammatory cytokines by blood monocytes was associated with increased amounts in plasma of the same cytokines, due to their in vivo release to the extracellular medium, we subsequently, investigated their plasma levels. Interestingly, increased IL1β, IL6, IL8, TNFα (P < 0.001) and IL10 (P = 0.024) levels were found in plasma of SM versus HD ( Figure 1C), both groups showing similarly low/undetectable IL12 levels ( Figure 1B in Supplementary Information). Of note, the same pattern of alteration was observed for soluble IL1β, IL6, IL8 and TNFα (P ≤ 0.006) across the distinct diagnostic subtypes of SM, although ISM patients displayed higher IL8 levels than BMM cases (P = 0.05), and increased IL10 levels were restricted to ISM and ASM patients (P = 0.010 and P = 0.015 vs. HD, respectively) ( Figure 2B). These data suggest that spontaneously increased cytokine production by circulating blood monocytes occurs in SM, which might lead to increased levels in plasma of the same inflammatory cytokines. In fact, a significant correlation between the number of IL1β + (P = 0.009), IL6 + (P = 0.043) and IL8 + (P = 0.016) blood monocytes and the plasma levels of the corresponding cytokines was observed ( Figure 1C in Supplementary Information).
In order to further assess whether increased spontaneous production of inflammatory cytokines, was associated with an overall enhanced cytokine production capacity after in vitro stimulation, blood monocytes from 7 SM patients and 15 HD were further stimulated in vitro with LPS plus IFNγ, prior to evaluation of cytokine production. Interestingly, in vitro stimulated blood monocytes from SM patients showed lower numbers of for example, IL1β + (P = 0.008) and TGFβ + monocytes (P = 0.013) than HD, although for the latter there were hardly any producing cells (Figure 3). These findings suggest that (compared to normal monocytes), circulating monocytes from SM patients are constitutively activated, but functionally exhausted. Alternatively, they might consist of different subsets which are for example, unresponsive to LPS and IFNγ (Figure 3 in Supplementary Information).

| DISCUSSION
Mast cells are key players in various physiologic and pathologic immune conditions due to their functional plasticity and the ability to release a broad array of bioactive mediators. 31 In line with these observations, previous studies in SM have recurrently reported abnormally high cytokine levels in plasma. 25,27,38,39 Interestingly, these have been repeatedly suggested  specifically recruits pDC to tissues) with potential consequences on activation of T-cells. 38 Interestingly, decreased counts of total DC, pDC and mDC (and their subsets) were more pronounced in ISM and ASM versus BMM, while Axl-DC were specifically increased in the latter patient group. At present, little is known about Axl-DC. Despite this, Axl-DC have been recently shown to promote T cell proliferation, and to be increased in inflammed tissues such as the lung and skin [46][47][48][49] Altogether, these data suggest that Axl DC might play a specific role in patients presenting with severe MC activation symptoms (e.g. anaphylaxis), a hallmark of BMM. In line with this hypothesis, our data also suggest a close association between higher counts of AXL DC counts, as well as virtually all subsets of DC and monocytes circulating in blood of SM patients, and the occurrence of anaphylaxis ( Table 4 in Supplementary Information).
In summary, here we confirm and extend on previous observations in SM, revealing an altered distribution of specific subsets of circulating monocytes and DC, associated with significantly increased numbers of activated but functionally exhausted blood monocytes, leading to increased levels of pro-inflammatory cytokines in plasma.